Substituted quinoxalines as kinase inhibitors

ABSTRACT

Disclosed herein are compounds of formula (I) and methods of inhibiting IKKβ and the NF-κB signaling and mTOR pathways.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/416,001, filed Nov. 22, 2010, and U.S. Provisional Application No.61/415,934, filed Nov. 22, 2010, each of which is incorporated byreference in its entirety.

STATEMENT OF U.S. GOVERNMENT SUPPORT

This invention was made with U.S. government support under Grant No. R01CA127239, awarded by the National Institutes of Health. The U.S.government has certain rights in the invention.

BACKGROUND

Since its discovery 25 years ago, NF-κB has been shown to regulate theexpression of over 200 immune, growth and inflammation genes. NF-κB isconstitutively active in proliferating T cells, B cells, thymocytes,monocytes and astrocytes. The clinically silent onset of PC has beenattributed to the upregulation of pro-inflammatory pathways such asNF-κB. NF-κB is constitutively active in most tumor cell lines and manytumor tissues derived from patients, but not in normal tissues. Asimilar observation was made in PC cell lines and pancreaticadenocarcinoma which showed constitutively activated RelA (p65 subunitof NF-κB), but not in normal pancreatic tissues orimmortalized/non-tumorigenic pancreatic epithelial cells. Studies alsoshowed that PC cell lines had increased levels of NF-κB subunitscompared to non-malignant proliferating intestinal cells. Thesepreclinical observations extend to PC patients: (i) High expression ofRelA (NF-κB subunit p65) was observed in 64% of histologically orcytologically verified locally advanced unresectable and/or metastaticPC patients and (ii) this correlates with increased expression of NF-κBtarget genes and poor prognosis in this patient subgroup. Downregulationof NF-κB (RelA) using siRNA sensitizes a subset of PC cells andpancreatic tumors in nude mice to gemcitabine. Inhibiting constitutiveNF-κB activity suppressed growth, angiogenesis and metastasis of PC.These observations suggest that NF-κB driven pro-inflammatory pathwayslead to a subset of PC's and modulating the NF-κB activity is a viabletherapeutic strategy for this subgroup.

The activity of IκB kinase β (IKKβ) is regulated by multiplephosphorylation events. IKKβ, like other kinases has an activation loop.Phosphorylation of two serine residues on the loop leads to theactivation of IKKβ. IKKβ also has a stretch of serine residues at theC-terminus and IKKβ activation leads to auto-phosphorylation of theC-terminus serine residues. Unlike phosphorylation of the activationloop, phosphorylation of the C-terminal residues dampens kinaseactivity. Therefore, phosphorylation of the C-terminal serine residuesnot only makes IKKβ activation transient but also provides docking sitesfor phosphatases to dephosphorylate the serine residues on theactivation loop. This suggests that IKKβ could exist in at least fourdistinct states as defined by its phosphorylation status and the kinaseactivity. The activation loop phosphorylated form of IKKβ is found inabout 50% of surgical tumor specimens and in about 10% of normaltissues. Therefore, knowledge regarding the phosphorylation status ofIKKβ is important from a biomarker and therapeutic developmentperspective. The lack of antibodies specific to the various states ofIKKβ makes this a challenging problem.

A need exists for IKKβ inhibitors and methods of treating IKKβ-mediateddisorders.

SUMMARY

Disclosed herein are compounds of formula (I):

wherein each R¹ is independently heteroaryl, aryl, or alkyl; L isselected from —NHC(O)NH—, —NH—SO₂—, —NHC(O)CH₂—, —NHC(S)NH—,

X is halo, alkyl, alkoxy, aryl, CO₂alkyl, COalkyl, or haloalkyl; R² isH, alkyl, alkoxy, CO₂alkyl, COalkyl, or haloalkyl; and R³ is H, alkyl,or alkoxy; or a salt, hydrate, or solvate thereof. In some cases, atleast one R¹ is furanyl, and in more specific cases, each R¹ is furanyl.In some cases, at least one R¹ is thiophenyl, and in more specificcases, each R¹ is thiophenyl. In some cases, at least one R¹ is phenyl,and in more specific cases, each R¹ is phenyl. In some cases, at leastone R¹ is pyridyl, and in more specific cases, each R¹ is pyridyl. Insome cases, at least one R¹ is alkyl, and in more specific cases, eachR¹ is alkyl, and in even more specific cases, the alkyl is selected frommethyl, ethyl, propyl, or isopropyl. In various cases, L is —NHC(O)NH—.In various cases, L is —NH—SO₂—. In various cases, L is —NHC(O)CH₂—. Invarious cases, L is —NHC(S)NH—. In various cases, L is

In various cases, L is

In some cases, X is halo, and in more specific cases is Br. In somecases, X is alkyl, and in more specific cases is methyl, ethyl, propyl,or isopropyl. In some cases, X is alkoxyl, and in more specific cases ismethoxyl, ethoxyl, propoxyl, or isopropoxyl, or even more specificcases, is methoxy or ethoxy. In some cases, X is CO₂alkyl, and in morespecific cases is CO₂CH₃ or CO₂CH₂CH₃. In some cases, X is COalkyl, andin more specific cases is COCH₃ or COCH₂CH₃. In some cases, X ishaloalkyl, and in more specific cases is CF₃, CHF₂, CH₂F, CCl₃, CHCl₂,CH₂Cl, CBr₃, CHBr₂, or CH₂Br. In various cases, R² is H. In variouscases, R² is alkyl, and in more specific cases is methyl, ethyl, propyl,or isopropyl. In various cases, R² is CO₂alkyl, and in more specificcases is CO₂CH₃ or CO₂CH₂CH₃. In various cases, R² is haloalkyl, and inmore specific cases is CF₃, CHF₂, CH₂F, CCl₃, CHCl₂, CH₂Cl, CBr₃, CHBr₂,or CH₂Br. In some cases, R³ is H. In some cases R³ is alkyl, and in morespecific cases, is methyl, ethyl, propyl, or isopropyl. In some cases,R³ is alkoxy, and in more specific cases, is methoxyl, ethoxyl,propoxyl, or isopropoxyl, or even more specific cases, is methoxy orethoxy.

Further disclosed herein are compositions comprising a compound asdisclosed herein and a pharmaceutically acceptable carrier.

Also disclosed herein are methods comprising contacting a cell with acompound disclosed herein in an amount effective to decrease activity ofIKKβ. In various cases, the contacting is in vitro. In other cases, thecontacting is in vivo. In various cases, the IKKβ is ahyperphosphorylated form of IKKβ.

Further disclosed herein are methods of inhibiting NFκB or mTORsignaling pathway comprising contacting a cell with a compound asdisclosed herein in an amount effective to inhibit the NFκB or mTORsignaling pathway.

Also disclosed herein are methods of treating a subject suffering froman IKKβ-dependent condition comprising administering to the subject atherapeutically effective amount of a compound as disclosed herein. Invarious cases, the condition is cancer, and in more specific cases, thecancer is pancreatic cancer, lymphoma, leukemia, colon cancer,colorectal cancer, familial adenomatous polyposis (FAP), hereditarynon-polyposis cancer (HNPCC), colitis-associated cancer, gastric cancer,or breast cancer. In some cases, the condition is diabetes. In variouscases, the condition is an inflammatory disease, and in more specificcases, is of rheumatoid arthritis, osteoarthritis, atherosclerosis,multiple sclerosis, chronic inflammatory demyelinatingpolyradiculoneuritis, asthma, inflammatory bowel disease, helicobacterpylori-associated gastritis, Crohn's disease, ulcerative colitis, orsystemic inflammatory response syndrome. In some cases, the condition isa neurological disease, and in more specific cases, is of ischemicstroke, traumatic brain injury, seizure, and a neurodegenerativedisorder, or even more specifically, Alzheimer's Disease, Parkinson'sDisease, ALS, or Huntington's. In any one of the methods disclosedherein, the compound disclosed herein can be administered in addition toa second therapeutic.

Further disclosed herein are electroluminescent devices comprising acompound as disclosed herein. In some specific cases, the compound has aformula (IA) or (IB):

wherein R¹ is 2-pyridyl, 3-pyridyl, 4-pyridyl, or thiophenyl. Alsodisclosed herein are organic electroluminescent devices which comprise acathode, an anode, and an organic thin film layer comprising at leastone layer sandwiched between the cathode and the anode, wherein the atleast one layer comprises a compound as disclosed herein, wherein thecompound has a blue fluorescence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. (A) Subset of a focused library screened to identify inhibitorsof TNF-α driven NF-κB activation. (B) Representative follow-up doseresponse curve with 13-197. (C) Representative blot showing theinhibition of TNF-α mediated NF-κB nuclear translocation by 13-197.

FIG. 1. (D) The NF-κB pathway was activated by TNF-α (20 ng/mL) in cellstreated with and without 13-197 (20 μM). Cell lysates were generated atindicated times and probed for p-IκBα, total-IκBα and tubulin.

FIG. 1. (E) Cell lysates were generated at the indicated times postTNF-α (20 ng/mL) stimulation with and without 13-197 (20 μM). Thelysates were subjected to reciprocal IP-IB with NF-κB (p65) and IκBαantibodies.

FIG. 1. (F) An ELISA assay to probe the effect of 13-197 on the kinaseactivity of full length IKKβ (n=2).

FIG. 1. (G) Proposed model and potential target of 13-197.

FIG. 2. (A) Pancreatic cancer cell lines were treated with 20 μM 13-197for 2 h and the lysates were probed for p-IκBα and total IκBα. (B)(left) MiaPaCa2 cells were treated with 13-197 (0, 10 and 20 μM) for 2 hand at 20 μM (right) for the indicated time. (C) MiaPaCa2 cells weretreated with 20 μM 13-197 for 4 h and 24 h. The cell lysates were probedfor p-S6K and p-eIF4EBP1. (D) A model that suggests the molecular targetfor 13-197 is the constitutively active IKKβ.

FIG. 3. (A) Pancreatic cells were treated with 13-197 for 72 h. IC₅₀values determined through curve fitting (n=2). (B) MiaPaCa2 cellstreated with 11 μM of 13-197 and viability measured at indicated times.*P<0.05, **P<0.005, ***P<0.005. (C) MiaPaCa2 cells were treated with 11μM 13-197 for 24 h followed by cell cycle analyses (n=3). (D) Celllysates were probed for cell cycle markers. (E) Dose- and time-dependenteffects of 13-197 on the levels of anti apoptotic proteins Mcl-1,Bcl-xL, XIAP and Survivin in MiaPaCa2 cells. MiaPaCa2 cells were treatedwith 13-197 at the indicated doses 2 h (left panel). Cells were treatedwith 20 μM the cell lysates were generated probed at the indicated timepoints (right panel).

FIG. 3. (F) MiaPaCa2 cells were treated with 11 μM of 13-197. After 24 hthe cells were subjected to a live/dead assay (n=3). (G) Invasion ofcells through matrigel coated microporous polycarbonate membrane wasmeasured in the presence and absence of 13-197 (n=3). Transwell serumdriven migration of cells in the presence and absence of 13-197 wasmeasured after a 24 h incubation. (n=3). **P<0.005 and ***P<0.0005

FIG. 4. (A) Plasma concentration time profile of 13-197 after oraladministration in mice. 13-197 concentrations in plasma are expressed asmean values (±SEM) at the 7 sampling times (0.5, 1, 2, 8, 24, 48, and 72h; n=3). Non-compartmental pharmacokinetic parameters are summarized asan inset in the figure. (B) Biodistribution of 13-197 in kidney, brain,lung, spleen, liver, and heart following oral administration of 150mg/kg in mice (data is expressed as mean±SEM, n=3). The area under thecurve (AUC) for each tissue is described in the inset. (C) Nude micewith orthotopic pancreatic tumors were treated with 13-197 (150 mg/Kg)or vehicle orally daily for 30 days. (D) Table describes the number ofanimals with tumors and number of tumor nodules found in various organs(E) Ki67 staining as a measure of proliferation index in tumor tissue(F) CD31+ staining as a measure of microvessel density. (G) Inflammationand Necrosis scored (blind) by a pathologist in the tumor tissue (H)13-197 levels measured in the pancreas, liver and serum 18 h after thelast treatment (1) Liver enzymes (AST and ALT) measured at the end ofthe study, **P<0.05

FIG. 5 shows induction of caspase 3/7 activity by 7c (also referred tothroughout this disclosure as 13-197) and etoposide a knownchemotherapeutic agent in MDA-MB-231 breast cancer cells (A) and PC3prostate cancer cells (B).

FIG. 6 shows apoptosis studies in Hela cells: (A) Induction of caspase3/7 by 7c and etoposide. (B) PARD cleavage induced by 7c assessed byWestern blot analyses. (C) Mcl-1 dependent induction of apoptosis by 7c.

FIG. 7 shows the effect of 13-197 on the IkBα phosphorylation in thetherapy-resistant MCL cells.

FIG. 8 shows effect of 13-197 on the down-regulation of the NF-κB (p65)phosphorylation and their nuclear translocation in the therapy-resistantMCL cell lines.

FIG. 9 (top) shows the effect of 13-197 on the down-regulation ofantiapoptotic proteins and (bottom) down regulation of cyclin D1 (aNF-κB target) in therapy-resistant MCL cells.

FIG. 10 shows the effect of 13-197 on the phosphorylation of the mTORtarget molecules S6K and eIF4EBP1 in the therapy-resistant MCL cells.

FIG. 11 shows the effect of 13-197 on therapy-resistant MCL cellsgrowth/proliferation in vitro.

FIG. 12 shows the effect of 13-197 on MCL cells apoptosis.

FIG. 13 shows the cytomorphology of Wright-Giemsa stained MCL cellsfollowing treatment with 13-197.

DETAILED DESCRIPTION

Disclosed herein are compounds having a structure of formula (I) andmethods of inhibiting NF-κB, or more specifically inhibiting IKKβ, usinga compound as disclosed herein.

The compounds disclosed herein have a structure of formula (I):

wherein each R¹ is independently heteroaryl, aryl, or alkyl; L isselected from —NHC(O)NH—, —NH—SO₂—, —NHC(O)CH₂—, —NHC(S)NH—,

X is halo, alkyl, alkoxy, CO₂alkyl, COalkyl, or haloalkyl; R² is H,alkyl, alkoxy, CO₂alkyl, COalkyl, or haloalkyl; or a salt, hydrate, orsolvate thereof.

In various embodiments disclosed herein, the compound of formula (I) haseach R¹ the same. In some specific embodiments disclosed herein, atleast one R¹ is furanyl or phenyl. In some specific embodimentsdisclosed herein, at least one R¹ is pyridyl or thiophenyl (alsoreferred to as thienyl). In some specific embodiments disclosed herein,at least one R¹ is alkyl. In various embodiments disclosed herein, L is

or —NHC(O)NH—. In various embodiments disclosed herein, X is Br. In somespecific embodiments disclosed herein, X is Br, OMe, CO₂Me, CF₃, orCOMe. In various embodiments disclosed herein, R² is H. In variousembodiments disclosed herein, R² is CF₃, OMe, or Me.

As used herein, “alkyl” refers to monovalent alkyl groups having 1 to 20carbon atoms. This term is exemplified by groups such as methyl, ethyl,propyl, isopropyl, butyl, sec-butyl, t-butyl, hexyl and the like. Linearand branched alkyls are included. The alkyl group can be a specificnumber of carbon atoms, as exemplified by the use of C_(x)-C_(y), wherex and y are integers. The compounds disclosed herein can have aC₄-C₂₀alkyl or more specifically a C₄-C₁₀alkyl substituent.

As used herein, the term “alkylene” refers to an alkyl group having asubstituent. For example, the term “alkylenearyl” refers to an alkylgroup substituted with an aryl group.

As used herein, the term “aryl” refers to a monocyclic or polycyclicaromatic group, preferably a monocyclic or bicyclic aromatic group,e.g., phenyl or naphthyl. Unless otherwise indicated, an aryl group canbe unsubstituted or substituted with one or more, and in particular oneto four groups independently selected from, for example, halo, alkyl,alkenyl, OCF₃, NO₂, CN, NC, OH, alkoxy, amino, CO₂H, CO₂alkyl, aryl, andheteroaryl. Exemplary aryl groups include, but are not limited to,phenyl, naphthyl, tetrahydronaphthyl, chlorophenyl, methylphenyl,methoxyphenyl, trifluoromethylphenyl, nitrophenyl,2,4-methoxychlorophenyl, and the like.

As used herein, the term “heteroaryl” refers to a monocyclic or bicyclicring system containing one or two aromatic rings and containing at leastone nitrogen, oxygen, or sulfur atom in an aromatic ring. Unlessotherwise indicated, a heteroaryl group can be unsubstituted orsubstituted with one or more, and in particular one to four,substituents selected from, for example, halo, alkyl, alkenyl, OCF₃,NO₂, CN, NC, OH, alkoxy, amino, CO₂H, CO₂alkyl, aryl, and heteroaryl. Insome cases, the heteroaryl group is substituted with one or more ofalkyl and alkoxy groups. Examples of heteroaryl groups include, but arenot limited to, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl,isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl,imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, andthiadiazolyl.

The term “alkoxy” used herein refers to an —Oalkyl group.

Asymmetric carbon atoms can be present. All such isomers, includingdiastereomers and enantiomers, as well as the mixtures thereof, areintended to be included in the scope of the disclosure herein. Incertain cases, compounds can exist in tautomeric forms. All tautomericforms are intended to be included in the scope of the disclosure herein.Likewise, when compounds contain an alkenyl or alkenylene group, thereexists the possibility of cis- and trans-isomeric forms of thecompounds. Both cis- and trans-isomers, as well as the mixtures of cis-and trans-isomers, are contemplated.

The salts, e.g., pharmaceutically acceptable salts, of the disclosedtherapeutics may be prepared by reacting the appropriate base or acidwith a stoichiometric equivalent of the therapeutic.

Acids commonly employed to form pharmaceutically acceptable saltsinclude inorganic acids such as hydrogen bisulfide, hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, aswell as organic acids such as para-toluenesulfonic acid, salicylic acid,tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylicacid, fumaric acid, gluconic acid, glucuronic acid, formic acid,glutamic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonicacid, carbonic acid, succinic acid, citric acid, benzoic acid and aceticacid, as well as related inorganic and organic acids. Suchpharmaceutically acceptable salts thus include anions, for examplesulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caprate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate,xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, O-hydroxybutyrate, glycolate, maleate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, and mandelate. In one embodiment,pharmaceutically acceptable acid addition salts include those formedwith mineral acids such as hydrochloric acid and hydrobromic acid, andespecially those formed with organic acids such as maleic acid.

Pharmaceutically acceptable base addition salts may be formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Pharmaceutically acceptable salts of compounds may also beprepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.Examples of metals used as cations are sodium, potassium, magnesium,ammonium, calcium, or ferric, and the like. Examples of suitable aminesinclude isopropylamine, trimethylamine, histidine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.

Similarly, pharmaceutically acceptable derivatives (e.g., esters),metabolites, hydrates, solvates and prodrugs of the therapeutic may beprepared by methods generally known to those skilled in the art. Thus,another embodiment provides compounds that are prodrugs of an activecompound. In general, a prodrug is a compound which is metabolized invivo (e.g., by a metabolic transformation such as deamination,dealkylation, de-esterification, and the like) to provide an activecompound. A “pharmaceutically acceptable prodrug” means a compound whichis, within the scope of sound medical judgment, suitable forpharmaceutical use in a patient without undue toxicity, irritation,allergic response, and the like, and effective for the intended use,including a pharmaceutically acceptable ester as well as a zwitterionicform, where possible, of the therapeutic. As used herein, the term“pharmaceutically acceptable ester” refers to esters that hydrolyze invivo and include those that break down readily in the human body toleave the parent compound or a salt thereof. Suitable ester groupsinclude, for example, those derived from pharmaceutically acceptablealiphatic carboxylic acids, particularly alkanoic, alkenoic,cycloalkanoic and alkanedioic acids, in which each alkyl or alkenylmoiety advantageously has not more than 6 carbon atoms. Representativeexamples of particular esters include, but are not limited to, formates,acetates, propionates, butyrates, acrylates and ethylsuccinates.Examples of pharmaceutically-acceptable prodrug types are described inHiguchi and Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of theA.C.S. Symposium Series, and in Roche, ed., Bioreversible Carriers inDrug Design, American Pharmaceutical Association and Pergamon Press,1987, both of which are incorporated herein by reference.

The compounds and compositions described herein may also includemetabolites. As used herein, the term “metabolite” means a product ofmetabolism of a compound of the embodiments or a pharmaceuticallyacceptable salt, analog, or derivative thereof, that exhibits a similaractivity in vitro or in vivo to a disclosed therapeutic. The compoundsand compositions described herein may also include hydrates andsolvates. As used herein, the term “solvate” refers to a complex formedby a solute (herein, the therapeutic) and a solvent. Such solvents forthe purpose of the embodiments preferably should not negativelyinterfere with the biological activity of the solute. Solvents may be,by way of example, water (e.g., forms a hydrate), ethanol, or aceticacid.

Some specific compounds disclosed herein include those shown in thebelow Scheme:

Specific compounds disclosed herein include

or a salt, hydrate or solvate thereof.

Other specific compounds contemplated include

or a salt, hydrate, or solvate thereof.

Still other specific compounds contemplated include

or a salt, hydrate or solvate thereof.

Yet other specific compounds are contemplated solely for use in thedisclosed therapeutic methods and/or organic electroluminescent devicesand methods described in detail below. These compounds are notcontemplated as compounds per se and are therefore specifically excludedfrom the genus of compounds of formula (I) described above. Thesecompounds include:

or a salt, hydrate, or solvate thereof.Therapeutic Methods

The present invention provides IKKβ inhibitors, as exemplified bycompounds of structural formula (I), for the treatment of a variety ofdiseases and conditions wherein inhibition of IKKβ has a beneficialeffect. In some cases, the compounds disclosed herein inhibit the NF-κBpathway and/or the mTOR pathway. In one embodiment, the presentinvention relates to a method of treating an individual suffering from adisease or condition wherein inhibition of IKKβ, NF-κB pathway and/orthe mTOR pathway provides a benefit comprising administering atherapeutically effective amount of a compound of structural formula (I)to an individual in need thereof.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to eliminating, reducing, or ameliorating a disease or condition,and/or symptoms associated therewith. Although not precluded, treating adisease or condition does not require that the disease, condition, orsymptoms associated therewith be completely eliminated. As used herein,the terms “treat,” “treating,” “treatment,” and the like may include“prophylactic treatment,” which refers to reducing the probability ofredeveloping a disease or condition, or of a recurrence of apreviously-controlled disease or condition, in a subject who does nothave, but is at risk of or is susceptible to, redeveloping a disease orcondition or a recurrence of the disease or condition. The term “treat”and synonyms contemplate administering a therapeutically effectiveamount of a compound of the invention to an individual in need of suchtreatment.

Within the meaning of the invention, “treatment” also includes relapseprophylaxis or phase prophylaxis, as well as the treatment of acute orchronic signs, symptoms and/or malfunctions. The treatment can beorientated symptomatically, for example, to suppress symptoms. It can beeffected over a short period, be oriented over a medium term, or can bea long-term treatment, for example within the context of a maintenancetherapy.

The compounds of structural formula (I) therefore can be used to treat avariety of diseases and conditions where modulation (e.g., inhibition oractivation) of IKKβ, NF-κB pathway and/or the mTOR pathway provides abenefit. Examples of such diseases and condition include, but are notlimited to cancer, diabetes, an inflammatory disease, and a neurologicaldisease.

The disclosed methods are useful for treating cancer, for example,inhibiting cancer growth, including complete cancer remission, forinhibiting cancer metastasis, and for promoting cancer resistance. Theterm “cancer growth” generally refers to any one of a number of indicesthat suggest change within the cancer to a more developed form. Thus,indices for measuring an inhibition of cancer growth include but are notlimited to a decrease in cancer cell survival, a decrease in tumorvolume or morphology (for example, as determined using computedtomographic (CT), sonography, or other imaging method), a delayed tumorgrowth, a destruction of tumor vasculature, improved performance indelayed hypersensitivity skin test, an increase in the activity ofcytolytic T-lymphocytes, and a decrease in levels of tumor-specificantigens.

The term “cancer resistance” refers to an improved capacity of a subjectto resist cancer growth, in particular growth of a cancer already had.In other words, the term “cancer resistance” refers to a decreasedpropensity for cancer growth in a subject.

In one aspect, the cancer comprises a solid tumor, for example, acarcinoma and a sarcoma. Carcinomas include malignant neoplasms derivedfrom epithelial cells which infiltrate, for example, invade, surroundingtissues and give rise to metastases. Adenocarcinomas are carcinomasderived from glandular tissue, or from tissues that form recognizableglandular structures. Another broad category of cancers includessarcomas and fibrosarcomas, which are tumors whose cells are embedded ina fibrillar or homogeneous substance, such as embryonic connectivetissue. The invention also provides methods of treatment of cancers ofmyeloid or lymphoid systems, including leukemias, lymphomas, and othercancers that typically are not present as a tumor mass, but aredistributed in the vascular or lymphoreticular systems. Furthercontemplated are methods for treatment of adult and pediatric oncology,growth of solid tumors/malignancies, myxoid and round cell carcinoma,locally advanced tumors, cancer metastases, including lymphaticmetastases. The cancers listed herein are not intended to be limiting.Age (child and adult), sex (male and female), primary and secondary,pre- and post-metastatic, acute and chronic, benign and malignant,anatomical location cancer embodiments and variations are contemplatedtargets. Cancers are grouped by embryonic origin (e.g., carcinoma,lymphomas, and sarcomas), by organ or physiological system, and bymiscellaneous grouping. Particular cancers may overlap in theirclassification, and their listing in one group does not exclude themfrom another.

Carcinomas that may targeted include adrenocortical, acinar, aciniccell, acinous, adenocystic, adenoid cystic, adenoid squamous cell,cancer adenomatosum, adenosquamous, adnexel, cancer of adrenal cortex,adrenocortical, aldosterone-producing, aldosterone-secreting, alveolar,alveolar cell, ameloblastic, ampullary, anaplastic cancer of thyroidgland, apocrine, basal cell, basal cell, alveolar, comedo basal cell,cystic basal cell, morphea-like basal cell, multicentric basal cell,nodulo-ulcerative basal cell, pigmented basal cell, sclerosing basalcell, superficial basal cell, basaloid, basosquamous cell, bile duct,extrahepatic bile duct, intrahepatic bile duct, bronchioalveolar,bronchiolar, bronchioloalveolar, bronchoalveolar, bronchoalveolar cell,bronchogenic, cerebriform, cholangiocelluarl, chorionic, choroidsplexus, clear cell, cloacogenic anal, colloid, comedo, corpus, cancer ofcorpus uteri, cortisol-producing, cribriform, cylindrical, cylindricalcell, duct, ductal, ductal cancer of the prostate, ductal cancer in situ(DCIS), eccrine, embryonal, cancer en cuirasse, endometrial, cancer ofendometrium, endometroid, epidermoid, cancer ex mixed tumor, cancer expleomorphic adenoma, exophytic, fibrolamellar, cancer fibro'sum,follicular cancer of thyroid gland, gastric, gelatinform, gelatinous,giant cell, giant cell cancer of thyroid gland, cancer gigantocellulare,glandular, granulose cell, hepatocellular, Hürthle cell, hypernephroid,infantile embryonal, islet cell carcinoma, inflammatory cancer of thebreast, cancer in situ, intraductal, intraepidermal, intraepithelial,juvenile embryonal, Kulchitsky-cell, large cell, leptomeningeal,lobular, infiltrating lobular, invasive lobular, lobular cancer in situ(LCIS), lymphoepithelial, cancer medullare, medullary, medullary cancerof thyroid gland, medullary thyroid, melanotic, meningeal, Merkel cell,metatypical cell, micropapillary, mucinous, cancer muciparum, cancermucocellulare, mucoepidermoid, cancer mucosum, mucous, nasopharyngeal,neuroendocrine cancer of the skin, noninfiltrating, non-small cell,non-small cell lung cancer (NSCLC), oat cell, cancer ossificans,osteoid, Paget's, papillary, papillary cancer of thyroid gland,periampullary, preinvasive, prickle cell, primary intrasseous, renalcell, scar, schistosomal bladder. Schneiderian, scirrhous, sebaceous,signet-ring cell, cancer simplex, small cell, small cell lung cancer(SCLC), spindle cell, cancer spongiosum, squamous, squamous cell,terminal duct, anaplastic thyroid, follicular thyroid, medullarythyroid, papillary thyroid, trabecular cancer of the skin, transitionalcell, tubular, undifferentiated cancer of thyroid gland, uterine corpus,verrucous, villous, cancer villosum, yolk sac, squamous cellparticularly of the head and neck, esophageal squamous cell, and oralcancers and carcinomas.

Sarcomas that may be targeted include adipose, alveolar soft part,ameloblastic, avian, botryoid, sarcoma botryoides, chicken,chloromatous, chondroblastic, clear cell sarcoma of kidney, embryonal,endometrial stromal, epithelioid, Ewing's, fascial, fibroblastic, fowl,giant cell, granulocytic, hemangioendothelial, Hodgkin's, idiopathicmultiple pigmented hemorrhagic, immunoblastic sarcoma of B cells,immunoblastic sarcoma of T cells, Jensen's, Kaposi's, kupffer cell,leukocytic, lymphatic, melanotic, mixed cell, multiple, lymphangio,idiopathic hemorrhagic, multipotential primary sarcoma of bone,osteoblastic, osteogenic, parosteal, polymorphous, pseudo-kaposi,reticulum cell, reticulum cell sarcoma of the brain, rhabdomyosarcoma,rots, soft tissue, spindle cell, synovial, telangiectatic, sarcoma(osteosarcoma)/malignant fibrous histiocytoma of bone, and soft tissuesarcomas.

Lymphomas that may be targeted include AIDS-related, non-Hodgkin's,Hodgkin's, T-cell, T-cell leukemia/lymphoma, African, B-cell, B-cellmonocytoid, bovine malignant, Burkitt's, centrocytic, lymphoma cutis,diffuse, diffuse, large cell, diffuse, mixed small and large cell,diffuse, small cleaved cell, follicular, follicular center cell,follicular, mixed small cleaved and large cell, follicular,predominantly large cell, follicular, predominantly small cleaved cell,giant follicle, giant follicular, granulomatous, histiocytic, largecell, immunoblastic, large cleaved cell, large nocleaved cell,Lennert's, lymphoblastic, lymphocytic, intermediate; lymphocytic,intermediately differentiated, plasmacytoid; poorly differentiatedlymphocytic, small lymphocytic, well differentiated lymphocytic,lymphoma of cattle; MALT, mantle cell, mantle zone, marginal zone,Mediterranean lymphoma mixed lymphocytic-histiocytic, nodular,plasmacytoid, pleomorphic, primary central nervous system, primaryeffusion, small b-cell, small cleaved cell, small concleaved cell,T-cell lymphomas; convoluted T-cell, cutaneous t-cell, small lymphocyticT-cell, undefined lymphoma, u-cell, undifferentiated, aids-related,central nervous system, cutaneous T-cell, effusion (body cavity based),thymic lymphoma, and cutaneous T cell lymphomas.

Leukemias and other blood cell malignancies that may be targeted includeacute lymphoblastic, acute myeloid, acute lymphocytic, acute myelogenousleukemia, chronic myelogenous, hairy cell, erythroleukemia,lymphoblastic, myeloid, lymphocytic, myelogenous, leukemia, hairy cell,T-cell, monocytic, myeloblastic, granulocytic, gross, hand mirror-cell,basophilic, hemoblastic, histiocytic, leukopenic, lymphatic,Schilling's, stem cell, myelomonocytic, monocytic, prolymphocytic,promyelocytic, micromyeloblastic, megakaryoblastic, megakaryoctyic,rieder cell, bovine, aleukemic, mast cell, myelocytic, plamsa cell,subleukemic, multiple myeloma, nonlymphocytic, chronic myelogenousleukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma,Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high gradeforms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chaindisease, myelodysplastic syndrome, and myelodysplasia and chronicmyelocytic leukemias.

Brain and central nervous system (CNS) cancers and tumors that may betargeted include astrocytomas (including cerebellar and cerebral), brainstem glioma, brain tumors, malignant gliomas, ependyrnoma, glioblastoma,medulloblastoma, supratentorial primitive neuroectodermal tumors, visualpathway and hypothalamic gliomas, primary central nervous systemlymphoma, ependymoma, brain stem glioma, visual pathway and hypothalamicglioma, extracranial germ cell tumor, medulloblastoma, myelodysplasticsyndromes, oligodendroglioma, myelodysplastic/myeloproliferativediseases, myelogenous leukemia, myeloid leukemia, multiple myeloma,myeloproliferative disorders, neuroblastoma, plasma cellneoplasm/multiple myeloma, central nervous system lymphoma, intrinsicbrain tumors, astrocytic brain tumors, gliomas, and metastatic tumorcell invasion in the central nervous system.

Gastrointestinal cancers that may be targeted include extrahepatic bileduct cancer, colon cancer, colon and rectum cancer, colorectal cancer,gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoidtumor, gastronintestinal carcinoid tumors, gastrointestinal stromaltumors, bladder cancers, islet cell carcinoma (endocrine pancreas),pancreatic cancer, islet cell pancreatic cancer, prostate cancer rectalcancer, salivary gland cancer, small intestine cancer, colon cancer, andpolyps associated with colorectal neoplasia.

Lung and respiratory cancers that may be targeted include bronchialadenomas/carcinoids, esophagus cancer esophageal cancer, esophagealcancer, hypopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer,lung carcinoid tumor, non-small cell lung cancer, small cell lungcancer, small cell carcinoma of the lungs, mesothelioma, nasal cavityand paranasal sinus cancer, nasopharyngeal cancer, nasopharyngealcancer, oral cancer, oral cavity and lip cancer, oropharyngeal cancer;paranasal sinus and nasal cavity cancer, and pleuropulmonary blastoma.

Urinary tract and reproductive cancers that may be targeted includecervical cancer, endometrial cancer, ovarian epithelial cancer,extragonadal germ cell tumor, extracranial germ cell tumor, extragonadalgerm cell tumor, ovarian germ cell tumor, gestational trophoblastictumor, spleen, kidney cancer, ovarian cancer, ovarian epithelial cancer,ovarian germ cell tumor, ovarian low malignant potential tumor, penilecancer, renal cell cancer (including carcinomas), renal cell cancer,renal pelvis and ureter (transitional cell cancer), transitional cellcancer of the renal pelvis and ureter, gestational trophoblastic tumor,testicular cancer, ureter and renal pelvis, transitional cell cancer,urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginalcancer, vulvar cancer, ovarian carcinoma, primary peritoneal epithelialneoplasms, cervical carcinoma, uterine cancer and solid tumors in theovarian follicle), superficial bladder tumors, invasive transitionalcell carcinoma of the bladder, and muscle-invasive bladder cancer.

Skin cancers and melanomas (as well as non-melanomas) that may betargeted include cutaneous t-cell lymphoma, intraocular melanoma, tumorprogression of human skin keratinocytes, basal cell carcinoma, andsquamous cell cancer. Liver cancers that may be targeted includeextrahepatic bile duct cancer, and hepatocellular cancers. Eye cancersthat may be targeted include intraocular melanoma, retinoblastoma, andintraocular melanoma Hormonal cancers that may be targeted include:parathyroid cancer, pineal and supratentorial primitive neuroectodermaltumors, pituitary tumor, thymoma and thymic carcinoma, thymoma, thymuscancer, thyroid cancer, cancer of the adrenal cortex, and ACTH-producingtumors.

Miscellaneous other cancers that may be targeted include advancedcancers, AIDS-related, anal cancer adrenal cortical, aplastic anemia,aniline, betel, buyo cheek, cerebriform, chimney-sweeps, clay pipe,colloid, contact, cystic, dendritic, cancer a deux, duct, dye workers,encephaloid, cancer en cuirasse, endometrial, endothelial, epithelial,glandular, cancer in situ, kang, kangri, latent, medullary, melanotic,mule-spinners', non-small cell lung, occult cancer, paraffin, pitchworkers', scar, schistosomal bladder, scirrhous, lymph node, small celllung, soft, soot, spindle cell, swamp, tar, and tubular cancers.

Miscellaneous other cancers that may be targeted also include carcinoid(gastrointestinal and bronchal) Castleman's disease chronicmyeloproliferative disorders, clear cell sarcoma of tendon sheaths,Ewing's family of tumors, head and neck cancer, lip and oral cavitycancer, Waldenström's macroglobulinemia, metastatic squamous neck cancerwith occult primary, multiple endocrine neoplasia syndrome, multiplemyeloma/plasma cell neoplasm, Wilms' tumor, mycosis fungoides,pheochromocytoma, sezary syndrome, supratentorial primitiveneuroectodermal tumors, unknown primary site, peritoneal effusion,malignant pleural effusion, trophoblastic neo-plasms, andhemangiopericytoma.

Specific cancers contemplated include acute lymphoblastic leukemia(ALL); acute myeloid leukemia (AML), adrenocortical carcinoma,AIDS-related cancer, Kaposi sarcoma, lymphoma, anal cancer, appendixcancer, astrocytomas, atypical teratoid/rhabdoid tumor, basal cellcarcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma,malignant fibrous histiocytoma, brain stem glioma, brain tumor,astrocytoma, brain and spinal cord tumor, brain stem glioma, CNSatypical teratoid/rhabdoid tumor, CNS embryonal tumor,craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma,medulloepithelioma, pineal parenchymal tumor, supratentorial primitiveneuroectodermal tumor, pineoblastoma, breast cancer, bronchial tumor,Burkitt lymphoma, non-Hodgkin lymphoma, carcinoid tumor, cervicalcancer, chordoma, chronic lymphocytic leukemia (CLL), chronicmyelogenous leukemia (CML), chronic myeloproliferative disorder, coloncancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma,embryonal tumor, endometrial cancer, ependymoblastoma, ependymoma,esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranialgerm cell tumor, extragonadal germ cell tumor, eye cancer, intraocularmelanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),gestational trophoblastic tumor, glioma, hairy cell leukemia, head andneck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis,langerhans cell. Hodgkin lymphoma, hypopharyngeal cancer, intraocularmelanoma, islet cell tumor, renal cell cancer, langerhans cellhistiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer,liver cancer, lobular carcinoma in situ (LCIS), lung cancer, lymphoma,macroglobulinemia, medulloblastoma, medulloepithelioma, melanoma, Merkelcell carcinoma, mesothelioma, metastatic squamous neck cancer, mouthcancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasmacell neoplasm, mycosis fungoide, myelodysplastic syndrome,myelodysplastic/myeloproliferative neoplasm, nasal cavity and paranasalsinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lungcancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreaticcancer, papillomatosis, paraganglioma, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor,pituitary tumor, plasma cell neoplasm, pleuropulomary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter transitional cell cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma,uterine sarcoma, soft tissue sarcoma, skin cancer, small cell lungcancer, small intestines cancer, squamous cell carcinoma, stomachcancer. T-cell lymphoma, testicular cancer, throat cancer, thymoma,thymic cancer, thyroid cancer, gestational trophoblastic cancer, vaginalcancer, vulvar cancer, Wilms tumor, and Waldenstrom macroglobulinemia.Specific cancers contemplated include pancreatic cancer, lymphoma,leukemia, colon cancer, colorectal cancer, familial adenomatouspolyposis (FAP), hereditary non-polyposis cancer (HNPCC),colitis-associated cancer, gastric cancer, and breast cancer. Specificinflammatory diseases contemplated include arthritis, rheumatoidarthritis, osteoarthritis, atherosclerosis, multiple sclerosis, chronicinflammatory demyelinating polyradiculoneuritis, asthma, inflammatorybowel disease, helicobacter pylori-associated gastritis, Crohn'sdisease, ulcerative colitis, and systemic inflammatory responsesyndrome.

The disclosed methods are useful for treating neurological disorders.For example, the NF-κB pathway is involved in various central nervoussystem (CNS) diseases such as ischemic stroke, traumatic brain injury,seizures, and neurodegenerative disorders. Non-limiting examples ofneurodegenerative disorders include Alzheimer's Disease, Parkinson'sDisease, ALS, and Huntington's. The role of the NF-κB pathway in CNSdisease is described in greater detail in Mattson et al., J. ClinicalInvest., 107(3):247 (2001).

A method of the present invention can be accomplished by administering acompound of structural formula (I) as the neat compound or as apharmaceutical composition. Administration of a pharmaceuticalcomposition, or neat compound of structural formula (I), can beperformed during or after the onset of the disease or condition ofinterest. Typically, the pharmaceutical compositions are sterile, andcontain no toxic, carcinogenic, or mutagenic compounds that would causean adverse reaction when administered. Further provided are kitscomprising a compound of structural formula (I) and, optionally, asecond therapeutic agent useful in the treatment of diseases andconditions wherein inhibition of IKKβ provides a benefit, packagedseparately or together, and an insert having instructions for usingthese active agents.

The methods disclosed herein also include the use of a compound orcompounds as described herein together with one or more additionaltherapeutic agents for the treatment of a disease. Thus, for example,the combination of active ingredients may be: (1) co-formulated andadministered or delivered simultaneously in a combined formulation; (2)delivered by alternation or in parallel as separate formulations; or (3)by any other combination therapy regimen known in the art. Whendelivered in alternation therapy, the methods described herein maycomprise administering or delivering the active ingredientssequentially, e.g., in separate solution, emulsion, suspension, tablets,pills or capsules, or by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e., serially, whereas insimultaneous therapy, effective dosages of two or more activeingredients are administered together. Various sequences of intermittentcombination therapy may also be used.

In some cases, a compound disclosed herein is administered and/orformulated with a second therapeutic, for example, a chemotherapeutic(e.g., an anti-cancer agent).

Chemotherapeutic agents contemplated for use include, withoutlimitation, alkylating agents including: nitrogen mustards, such asmechlor-ethamine, cyclophosphamide, ifosfamide, melphalan andchlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU),and semustine (methyl-CCNU); ethylenimines/methylmelamine such asthriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa),hexamethylmelamine (HMM, altretamine); alkyl sulfonates such asbusulfan; triazines such as dacarbazine (DTIC); antimetabolitesincluding folic acid analogs such as methotrexate and trimetrexate,pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine,gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine,2,2′-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine,6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin),erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products includingantimitotic drugs such as paclitaxel, vinca alkaloids includingvinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine,and estramustine phosphate; epipodophylotoxins such as etoposide andteniposide; antibiotics such as actinomycin D, daunomycin (rubidomycin),doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin(mithramycin), mitomycinC, and actinomycin; enzymes such asL-asparaginase; biological response modifiers such as interferon-alpha,IL-2, G-CSF and GM-CSF; miscellaneous agents including platinumcoordination complexes such as cisplatin and carboplatin,anthracenediones such as mitoxantrone, substituted urea such ashydroxyurea, methylhydrazine derivatives including N-methylhydrazine(MIH) and procarbazine, adrenocortical suppressants such as mitotane(o,p′-DDD) and aminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate; estrogen such as diethylstilbestrol and ethinyl estradiolequivalents; antiestrogen such as tamoxifen; androgens includingtestosterone propionate and fluoxymesterone/equivalents; antiandrogenssuch as flutamide, gonadotropin-releasing hormone analogs andleuprolide; non-steroidal antiandrogens such as flutamide; kinaseinhibitors, histone deacelylase inhibitors, methylation inhibitors,proteasome inhibitors, monoclonal antibodies, oxidants, anti-oxidants,telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, statinhibitors, and nanoparticles.

Synthesis of Compounds

The compounds disclosed herein can be synthesized through any meansavailable to the synthetic chemist and in view of the guidance of theschemes below. Non-limiting examples for preparing compounds disclosedherein is provided below.

Exemplary reagents and conditions for reactions of Scheme 1 are: (i)Ethanol, reflux, 36-48 h; (ii) Pd/C, H₂, ethanol, room temperature, 6-8h; (iii) R₂NCO, DIPEA, DCM 24-72 h; (iv) R₂COCl, DCM, 4 h; (v) TsCl,TEA, DCM 6 h; (vi) R₅NCS (2 eq), DCM, reflux; (vii) Triphosgene, DIPEA,DCM, 4 h; Amine, DCM, 8-24 h; (viii) R₆PhNCO (1.5 eq), DIPEA, DCM, 12-24h.

Exemplary compounds prepared by the synthetic scheme outlined in Scheme1 are listed in Table 1.

TABLE 1 Entry R₁ R₂ R₃ R₄ 5a Methyl −COCH₃ −H −H 5b Furanyl −COCH₃ −H −H5c Thienyl −COCH₃ −H −H 5d Phenyl −COCH₃ −H −H 5e Methyl −CONHPh −H −H5f Furanyl −CONHPh −H −H 5g Thienyl −CONHPh −H −H 5h Phenyl −CONHPh −H−H 5i Methyl −H −H −SO₂Tol 5j Furanyl −SO₂Tol −SO₂Tol −H 5k Thienyl−SO₂Tol −SO₂Tol −H 5l Phenyl −SO₂Tol −H −H R₁ R₅ 6a Furanyl −CS-Phenyl6b Thienyl −CS-Phenyl 6c Phenyl −CS-Phenyl 6d Furanyl−CS-(4-Nitro)-Phenyl 6e Thienyl −CS-(4-Nitro)-Phenyl 6f Phenyl−CS-(4-Nitro)-Phenyl 6g Methyl −CO-Pyrrolidine 6h Furanyl−CO-Pyrrolidine 6i Phenyl −CO-Pyrrolidine 6j Methyl−CO-(4-Benzyl)-Piperidine 6k Furanyl −CO-(4-Benzyl)-Piperidine 6l Methyl−CO-Piperdine 6m Furanyl −CO-Morpholinc R₁ R₆ 7a Furanyl −F 7b Furanyl−Cl 7c Furanyl −Br 7d Furanyl -Phenyl

The compounds were screened in a growth inhibition assay at 20 μM over a72 h period in a panel of cancer cell lines (lung-A549;pancreatic-Aspc1; colon-HT29; breast-MDAMB231; prostate-PC3;ovarian-SKOV3 and bone-U2OS) and the results are summarized in Table 2.In the 5 compound series only 5a, 5b and 5f inhibited growth of thevarious cancer cell lines. This suggests that the furan substitutions atthe 2,3-positions are clearly better than the other three. It alsosuggests that sufonamide substitution at the 6-position is not suitablefor the growth inhibitory activity. The screening results in the 6compound series again show that all the furan compounds (6a, 6d, 6h, 6kand 6m) were active. It also shows that substitution at the 4-positionof the phenyl thioureas plays a role in the biological activity (6b and6c vs. 6e and 6f). This size effect was also observed with the ureacompounds from the secondary amine (6j vs. 6l and 6k vs. 6m).

TABLE 2 % Growth inhibition at 20 μM MDA-MB- Entry A549 AsPC1 HT29 231PC3 SKOV3 U2OS 5a 23.4 ± 3.3 26.2 ± 2.8 31.7 ± 2.8 25.7 ± 5.1  21.8 ±15.1 4.0 ± 3.6 31.4 ± 3.6 5b 57.8 ± 7.0 Inactive  7.6 ± 5.1 20.3 ± 5.957.7 ± 9.4 9.0 ± 3.4 Inactive 5c Inactive Inactive Inactive InactiveInactive Inactive Inactive 5d Inactive Inactive Inactive InactiveInactive Inactive Inactive 5e Inactive Inactive Inactive InactiveInactive Inactive Inactive 5f 54.6 ± 2.6  9.6 ± 6.6  52.1 ± 11.6  31.3 ±10.7 70.6 ± 1.3 24.7 ± 1.1  59.6 ± 1.6 5g Inactive Inactive InactiveInactive Inactive Inactive Inactive 5h Inactive Inactive InactiveInactive Inactive Inactive Inactive 5i Inactive Inactive InactiveInactive Inactive Inactive Inactive 5j Inactive Inactive InactiveInactive Inactive Inactive Inactive 5k Inactive Inactive InactiveInactive Inactive Inactive Inactive 5l Inactive Inactive InactiveInactive Inactive Inactive Inactive 6a 18.0 ± 0.1 Inactive  26.1 ± 15.0 19.1 ± 12.5 56.8 ± 1.4 Inactive 21.4 ± 8.4 6b Inactive InactiveInactive Inactive Inactive Inactive Inactive 6c Inactive InactiveInactive Inactive Inactive Inactive Inactive 6d 19.2 ± 8.9 Inactive 42.0± 1.9  9.0 ± 4.7 43.4 ± 9.3 Inactive 32.2 ± 0.1 6e Inactive Inactive 23.9 ± 27.8 53.3 ± 7.2  19.5 ± 37.4 Inactive 50.6 ± 3.9 6f  16.6 ± 11.2 55.5 ± 25.4 81.2 ± 0.7 72.6 ± 0.7 81.0 ± 8.5 55.8 ± 30.7 76.1 ± 0.4 6gInactive Inactive Inactive Inactive Inactive Inactive Inactive 6h 23.3 ±2.9 11.6 ± 4.2 16.7 ± 3.7 18.2 ± 7.0 53.1 ± 9.9 16.9 ± 4.9  46.5 ± 4.46i 11.0 ± 1.3 29.4 ± 9.8 Inactive 16.7 ± 3.9 56.9 ± 2.7 18.5 ± 4.3  54.2± 2.2 6j 40.3 ± 1.9 Inactive  16.3 ± 10.1 12.4 ± 5.0  30.6 ± 11.6 8.9 ±5.1 33.5 ± 0.4 6k 93.1 ± 7.8 >100 55.2 ± 4.1 90.8 ± 4.5 >100 47.6 ± 2.5 80.3 ± 2.4 6l Inactive Inactive Inactive Inactive Inactive InactiveInactive 6m  39.7 ± 12.8 Inactive 19.4 ± 3.6 10.9 ± 5.0 48.9 ± 7.5Inactive Inactive 7a >100 >100 74.2 ± 5.2 38.4 ± 2.7 90.7 ± 4.1 Inactive55.6 ± 6.6 7b 99.2 ± 5.2 81.1 ± 2.0 86.3 ± 7.7 92.5 ± 6.6 86.4 ± 1.155.8 ± 24.1 87.4 ± 1.1 7c >100 >100 88.6 ± 4.6 >100 >100 94.9 ±7.9  >100 7d 13.8 ± 9.8 Inactive 72.4 ± 7.6 50.2 ± 8.9 88.5 ± 1.9Inactive 46.9 ± 7.4

These results prompted us to synthesize, four additional compounds toprobe the size effect at the 4-position on a phenyl urea (7a-d).Evaluation of these analogs in the growth inhibition assay clearlyshowed a size effect and compound 7c with bromo substitution at the4-position was identified as the best compound. A dose-response studywith compound 7c shows low-μM GI₅₀ values against a panel of cancer celllines (Table 3). In summary this iterative synthesis and screeningeffort show that the furan substitution at the 2,3-position, a urea atthe 6-position and the substitutent at the para-position of a phenylurea are important for the biological activity. These studies alsoresulted in the identification 7c with low-μM GI₅₀ values against apanel of cancer cell lines.

TABLE 3 Cell line GI₅₀ (μM) A549 6.4 ± 3.0 AsPC1 17.3 ± 0.9  HT29 12.1 ±7.4  MDA-MB-231 8.4 ± 0.9 PC3 5.9 ± 2.7 SKOV3 16.8 ± 5.2  U2OS 10.8 ±0.2 

Caspases are a class of cysteine proteinases that are activated duringapoptosis and measuring caspase activity is often used to detectactivation of apoptotic signaling. To determine if the growth inhibitoryeffects observed with 7c in various cancer cell lines were a result ofprogrammed cell death, the ability of 7c to induce caspase-3/7 wasexplored. The results show that 7c induces caspase 3/7 much more rapidlycompared to the positive control (Etoposide) in MDA-MB-231 and PC3 cells(FIGS. 5A and B) and the induction is sustained for 72 h in these celllines.

Bcl-xL, Bcl-2 and Mcl-1 are antiapoptotic proteins that are implicatedin the survival of cancer cells. Bad3SA is the endogenous inhibitor ofBcl-xL and Bcl-2 but not Mcl-1. Using Hela cells that over expressBad3SA, we explored the mechanistic basis for the induction of apoptosisby 7c. In these cell lines expression of Bad3SA is under the control ofDoxcycline (Dox). The apoptosis studies carried out in these cell linesare summarized in FIG. 6. As with the other cancer cell lines we observeinduction of caspase 3/7 and PARP cleavage by 7c (FIGS. 6A and 6Brespectively). We next carried out a dose response study with 7c incells treated with Dox (1 μg/mL for 3 h) to induce Bad3SA [(+) Dox] anduntreated cells [(−) Dox]. The cells were incubated with 7c and apositive control (DNA damaging agent Camptothecin, CPT) for 12 h. Celldeath was measured by counting the number of condensed nuclei. A dosedependent increase in the induction of apoptosis in the (+) Dox cellswas observed, indicating that 7c induces apoptosis in a Mcl-1 dependentmanner (FIG. 6C).

In summary, a focused library of 2,3-substituted quinoxalin-6-amineanalogs was synthesized and evaluated in a panel of cancer cell linesfor growth inhibition. The preliminary structure activity relationship(SAR) showed bis-furan substitution at the 2,3-positions was favored. Acomparison of a series of linkers between the 2,3-disubstitutedquinoxaline and a substituted phenyl ring showed that a urea linker wasoptimal for the antiproliferative activity. In addition, the size of thesubstituent at the 4-position of the phenyl ring was important for theactivity. These led to the identification of bisfuranylquinoxalineureaanalog (7c) with low micromolar potency against the panel of cancer celllines. The analog 7c induces caspase 3/7 activation, PARP cleavage andMcl-1 dependent apoptosis.

Dosing and Pharmaceutical Formulations

The term “therapeutically effective amount,” as used herein, refers toan amount of a compound sufficient to treat, ameliorate, or prevent theidentified disease or condition, or to exhibit a detectable therapeutic,prophylactic, or inhibitory effect. The effect can be detected by, forexample, an improvement in clinical condition, reduction in symptoms, orby any of the assays or clinical diagnostic tests described herein orknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Therapeutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician.

Dosages of the therapeutic can alternately be administered as a dosemeasured in mg/kg. Contemplated mg/kg doses of the disclosedtherapeutics include about 0.001 mg/kg to about 1000 mg/kg. Specificranges of doses in mg/kg include about 0.1 mg/kg to about 500 mg/kg,about 0.5 mg/kg to about 200 mg/kg, about 1 mg/kg to about 100 mg/kg,about 2 mg/kg to about 50 mg/kg, and about 5 mg/kg to about 30 mg/kg.

A compound of structural formula (I) used in a method of the presentinvention can be administered in an amount of about 0.005 to about 750milligrams per dose, about 0.05 to about 500 milligrams per dose, orabout 0.5 to about 250 milligrams per dose. For example, a compound ofstructural formula (I) can be administered, per dose, in an amount ofabout 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, or 750 milligrams, including alldoses between 0.005 and 750 milligrams.

As herein, the compounds described herein may be formulated inpharmaceutical compositions with a pharmaceutically acceptableexcipient, carrier, or diluent. The compound or composition comprisingthe compound is administered by any route that permits treatment of thedisease or condition. One route of administration is oraladministration. Additionally, the compound or composition comprising thecompound may be delivered to a patient using any standard route ofadministration, including parenterally, such as intravenously,intraperitoneally, intrapulmonary, subcutaneously or intramuscularly,intrathecally, topically, transdermally, rectally, orally, nasally or byinhalation. Slow release formulations may also be prepared from theagents described herein in order to achieve a controlled release of theactive agent in contact with the body fluids in the gastro intestinaltract, and to provide a substantial constant and effective level of theactive agent in the blood plasma. The crystal form may be embedded forthis purpose in a polymer matrix of a biological degradable polymer, awater-soluble polymer or a mixture of both, and optionally suitablesurfactants. Embedding can mean in this context the incorporation ofmicro-particles in a matrix of polymers. Controlled release formulationsare also obtained through encapsulation of dispersed micro-particles oremulsified micro-droplets via known dispersion or emulsion coatingtechnologies.

Administration may take the form of single dose administration, or acompound as disclosed herein can be administered over a period of time,either in divided doses or in a continuous-release formulation oradministration method (e.g., a pump). However the compounds of theembodiments are administered to the subject, the amounts of compoundadministered and the route of administration chosen should be selectedto permit efficacious treatment of the disease condition.

In an embodiment, the pharmaceutical compositions are formulated withone or more pharmaceutically acceptable excipient, such as carriers,solvents, stabilizers, adjuvants, diluents, etc., depending upon theparticular mode of administration and dosage form. The pharmaceuticalcompositions should generally be formulated to achieve a physiologicallycompatible pH, and may range from a pH of about 3 to a pH of about 11,preferably about pH 3 to about pH 7, depending on the formulation androute of administration. In alternative embodiments, the pH is adjustedto a range from about pH 5.0 to about pH 8. More particularly, thepharmaceutical compositions may comprise a therapeutically orprophylactically effective amount of at least one compound as describedherein, together with one or more pharmaceutically acceptableexcipients. Optionally, the pharmaceutical compositions may comprise acombination of the compounds described herein, or may include a secondactive ingredient useful in the treatment or prevention of a disorder asdisclosed herein (e.g., an anticancer agent or an anti-inflammatoryagent).

Formulations, e.g., for parenteral or oral administration, are mosttypically solids, liquid solutions, emulsions or suspensions, whileinhalable formulations for pulmonary administration are generallyliquids or powders. A pharmaceutical composition can also be formulatedas a lyophilized solid that is reconstituted with a physiologicallycompatible solvent prior to administration. Alternative pharmaceuticalcompositions may be formulated as syrups, creams, ointments, tablets,and the like.

The term “pharmaceutically acceptable excipient” refers to an excipientfor administration of a pharmaceutical agent, such as the compoundsdescribed herein. The term refers to any pharmaceutical excipient thatmay be administered without undue toxicity.

Pharmaceutically acceptable excipients are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there exists awide variety of suitable formulations of pharmaceutical compositions(see, e.g., Remington's Pharmaceutical Sciences).

Suitable excipients may be carrier molecules that include large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and inactive virus particles. Other exemplary excipients includeantioxidants (e.g., ascorbic acid), chelating agents (e.g., EDTA),carbohydrates (e.g., dextrin, hydroxyalkylcellulose, and/orhydroxyalkylmethylcellulose), stearic acid, liquids (e.g., oils, water,saline, glycerol and/or ethanol) wetting or emulsifying agents, pHbuffering substances, and the like. Liposomes are also included withinthe definition of pharmaceutically acceptable excipients.

The pharmaceutical compositions described herein are formulated in anyform suitable for an intended method of administration. When intendedfor oral use for example, tablets, troches, lozenges, aqueous or oilsuspensions, non-aqueous solutions, dispersible powders or granules(including micronized particles or nanoparticles), emulsions, hard orsoft capsules, syrups or elixirs may be prepared. Compositions intendedfor oral use may be prepared according to any method known to the artfor the manufacture of pharmaceutical compositions, and suchcompositions may contain one or more agents including sweetening agents,flavoring agents, coloring agents and preserving agents, in order toprovide a palatable preparation.

Pharmaceutically acceptable excipients particularly suitable for use inconjunction with tablets include, for example, inert diluents, such ascelluloses, calcium or sodium carbonate, lactose, calcium or sodiumphosphate; disintegrating agents, such as cross-linked povidone, maizestarch, or alginic acid; binding agents, such as povidone, starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc.

Tablets may be uncoated or may be coated by known techniques includingmicroencapsulation to delay disintegration and adsorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample celluloses, lactose, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with non-aqueousor oil medium, such as glycerin, propylene glycol, polyethylene glycol,peanut oil, liquid paraffin or olive oil.

In another embodiment, pharmaceutical compositions may be formulated assuspensions comprising a compound of the embodiments in admixture withat least one pharmaceutically acceptable excipient suitable for themanufacture of a suspension.

In yet another embodiment, pharmaceutical compositions may be formulatedas dispersible powders and granules suitable for preparation of asuspension by the addition of suitable excipients.

Excipients suitable for use in connection with suspensions includesuspending agents (e.g., sodium carboxymethylcellulose, methylcellulose,hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone,gum tragacanth, gum acacia); dispersing or wetting agents (e.g., anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethyleneoxycethanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate)); andthickening agents (e.g., carbomer, beeswax, hard paraffin or cetylalcohol). The suspensions may also contain one or more preservatives(e.g., acetic acid, methyl or n-propyl p-hydroxy-benzoate); one or morecoloring agents; one or more flavoring agents; and one or moresweetening agents such as sucrose or saccharin.

The pharmaceutical compositions may also be in the form of oil-in wateremulsions. The oily phase may be a vegetable oil, such as olive oil orarachis oil, a mineral oil, such as liquid paraffin, or a mixture ofthese. Suitable emulsifying agents include naturally-occurring gums,such as gum acacia and gum tragacanth; naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids; hexitol anhydrides, such as sorbitan monooleate; and condensationproducts of these partial esters with ethylene oxide, such aspolyoxyethylene sorbitan monooleate. The emulsion may also containsweetening and flavoring agents. Syrups and elixirs may be formulatedwith sweetening agents, such as glycerol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative, a flavoringor a coloring agent.

Additionally, the pharmaceutical compositions may be in the form of asterile injectable preparation, such as a sterile injectable aqueousemulsion or oleaginous suspension. This emulsion or suspension may beformulated by a person of ordinary skill in the art using those suitabledispersing or wetting agents and suspending agents, including thosementioned above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, such as a solution in 1,2-propane-diol.

The sterile injectable preparation may also be prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile fixed oils may be employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids (e.g., oleicacid) may likewise be used in the preparation of injectables.

To obtain a stable water-soluble dose form of a pharmaceuticalcomposition, a pharmaceutically acceptable salt of a compound describedherein may be dissolved in an aqueous solution of an organic orinorganic acid, such as 0.3 M solution of succinic acid, or morepreferably, citric acid. If a soluble salt form is not available, thecompound may be dissolved in a suitable co-solvent or combination ofco-solvents. Examples of suitable co-solvents include alcohol, propyleneglycol, polyethylene glycol 300, polysorbate 80, glycerin and the likein concentrations ranging from about 0 to about 60% of the total volume.In one embodiment, the active compound is dissolved in DMSO and dilutedwith water.

The pharmaceutical composition may also be in the form of a solution ofa salt form of the active ingredient in an appropriate aqueous vehicle,such as water or isotonic saline or dextrose solution. Also contemplatedare compounds which have been modified by substitutions or additions ofchemical or biochemical moieties which make them more suitable fordelivery (e.g., increase solubility, bioactivity, palatability, decreaseadverse reactions, etc.), for example by esterification, glycosylation,PEGylation, etc.

In some embodiments, the compounds described herein may be formulatedfor oral administration in a lipid-based formulation suitable for lowsolubility compounds. Lipid-based formulations can generally enhance theoral bioavailability of such compounds.

As such, pharmaceutical compositions comprise a therapeutically orprophylactically effective amount of a compound described herein,together with at least one pharmaceutically acceptable excipientselected from the group consisting of medium chain fatty acids andpropylene glycol esters thereof (e.g., propylene glycol esters of ediblefatty acids, such as caprylic and capric fatty acids) andpharmaceutically acceptable surfactants, such as polyoxyl 40hydrogenated castor oil.

In some embodiments, cyclodextrins may be added as aqueous solubilityenhancers. Exemplary cyclodextrins include hydroxypropyl, hydroxyethyl,glucosyl, maltosyl and maltotriosyl derivatives of α-, β-, andγ-cyclodextrin. A specific cyclodextrin solubility enhancer ishydroxypropyl-o-cyclodextrin (BPBC), which may be added to any of theabove-described compositions to further improve the aqueous solubilitycharacteristics of the compounds of the embodiments. In one embodiment,the composition comprises about 0.1% to about 20%hydroxypropyl-o-cyclodextrin, more preferably about 1% to about 15%hydroxypropyl-o-cyclodextrin, and even more preferably from about 2.5%to about 10% hydroxypropyl-o-cyclodextrin. The amount of solubilityenhancer employed will depend on the amount of the compound of theinvention in the composition.

Organic Electroluminescence

Compounds disclosed herein can be blue fluorescent. In particular,compounds wherein at least one R¹ is pyridyl (e.g., 2-, 3-, or4-pyridyl) or thiophenyl exhibit a blue fluorescent property. Morespecifically, compounds having a structure of formula (IA) or (IB) canbe blue fluorescent:

wherein R¹ is 2-pyridyl, 3-pyridyl, 4-pyridyl, or thiophenyl, and R², L,and X are as defined above.

Thus, compounds disclosed herein can be used as organicelectroluminescent (EL) materials and devices. The light emission of theorganic EL device is a phenomenon in which, when an electric field isapplied across the two electrodes, electrons are injected from thecathode side and holes are injected from the anode side, the electronsrecombine with the holes in the light emitting layer to induce anexcited state, and then, when the excited state returns to the originalstate, it emits energy as light.

The organic EL device disclosed herein comprises an anode, a cathode andan organic thin film layer comprising at least one layer sandwichedbetween the anode and the cathode, wherein at least one layer in theorganic thin film layer comprises a compound having a structure offormula (I).

The organic EL device disclosed herein emits bluish light, due to anenergy gap. The material for organic EL devices can be a host materialof the organic EL device. The host material is a material into whichholes and electrons can be injected and which has the function oftransporting holes and electrons and emitting fluorescent light byrecombination of holes and electrons.

In the light emitting layer of the organic EL device, in general, thesinglet exciton and the triplet exciton are contained in the formedexcited molecules as a mixture, and it is reported that the tripletexciton is formed in a greater amount such that the ratio of the amountof the singlet exciton to the amount of the triplet exciton is 1:3. Inconventional organic EL devices using the phosphorescence, the excitoncontributing to the light emission is the singlet exciton, and thetriplet exciton does not emit light. Therefore, the triplet exciton isultimately consumed as heat, and the light is emitted by the singletexciton which is formed in a smaller amount. Therefore, in these organicEL devices, the energy transferred to the triplet exciton causes a greatloss in the energy generated by the recombination of holes andelectrons.

In some embodiments, by using the material as disclosed herein for thephosphorescence device, the efficiency of light emission three times asgreat as that of a device using fluorescence can be obtained since thetriplet exciton can be used for the emission of light. Further, invarious embodiments, when the compound as disclosed herein is used forthe light emitting layer of the phosphorescence device, an excitedtriplet level in an energy state higher than the excited triplet levelof a phosphorescent organometallic complex comprising a metal selectedfrom the Group 7 to 11 of the Periodic Table contained in the layer, isachieved; the film having a more stable form is provided; the glasstransition temperature is higher (Tg: 80 to 160° C.); holes and/orelectrons are efficiently transported; the compound is electrochemicallyand chemically stable; and the formation of impurities which may work asa trap or cause loss in the light emission is suppressed during thepreparation and the use.

The organic EL device disclosed herein comprises a cathode, an anode andan organic thin film layer comprising at least one layer and sandwichedbetween the cathode and the anode. When the organic thin film layercomprises a single layer, a light emitting layer is formed between theanode and the cathode. The light emitting layer contains a lightemitting material and may further contain a hole injecting material fortransporting holes injected from the anode to the light emittingmaterial or an electron injecting material for transporting electronsinjected from the cathode to the light emitting material. In some cases,the light emitting material exhibits a high quantum efficiency offluorescence, is able to transport both holes and electrons and/or formsa uniform thin layer. Examples of the organic EL device of themulti-layer type include organic EL devices comprising a laminate havinga multi-layer construction such as (the anode/the hole injectinglayer/the light emitting layer/the cathode), (the anode/the lightemitting layer/the electron injecting layer/the cathode) and (theanode/the hole injecting layer/the light emitting layer/the electroninjecting layer/the cathode).

For the light emitting layer, in addition to the compounds disclosedherein, conventional host materials, light emitting materials, dopingmaterials, hole injecting materials and electron injecting materials andcombinations of these materials can be used in combination. By using amulti-layer structure for the organic EL device, decreases in theluminance and the life due to quenching can be prevented, and theluminance of emitted light and the efficiency of light emission can beimproved with other doping materials. By using other doping materialscontributing to the light emission of the phosphorescence incombination, the luminance of emitted light and the efficiency of lightemission can be improved in comparison with those of conventionaldevices.

In the organic EL device disclosed herein, the hole injecting layer, thelight emitting layer and the electron injecting layer may each have amulti-layer structure. When the hole injecting layer has a multi-layerstructure, the layer into which holes are injected from the electrode iscalled as a hole injecting layer, and the layer which receives holesfrom the hole injecting layer and transports holes to the light emittinglayer is called as a hole transporting layer. Similarly, when theelectron injecting layer has a multi-layer structure, the layer intowhich electron are injected from the electrode is called as an electroninjecting layer, and the layer which receives electrons from theelectron injecting layer and transports electrons to the light emittinglayer is called as an electron transporting layer. The layers areselected in accordance with the energy levels of the material, heatresistance and adhesion with the organic thin film layers or the metalelectrodes.

In the organic EL device disclosed herein, the electron transportinglayer and the hole transporting layer can contain the material fororganic EL devices of the present invention which comprises the compoundrepresented by general formula (I).

Examples of the light emitting material and the host material which canbe used for the organic thin film layer in combination with the compoundrepresented by general formula (I) include anthracene, naphthalene,phenanthrene, pyrene, tetracene, coronene, chrysene, fluoresceine,perylene, phthaloperylene, naphthaloperylene, perynone, phthaloperynone,naphthaloperynone, diphenylbutadiene, tetraphenyl-butadiene, coumarine,oxadiazole, aldazine, bisbenzoxazoline, bisstyryl, pyrazine,cyclopentadiene, metal complexes of quinoline, metal complexes ofaminoquinoline, metal complexes of benzoquinoline, imines,diphenylethylene, vinylanthracene, diaminoanthracene, diaminocarbazole,pyrane, thiopyrane, polymethine, melocyanine, oxinoid compounds chelatedwith imidazole, quinacridone, rubrene, stilbene-based derivatives andfluorescent pigments. However, the light emitting material and the hostmaterial are not limited to the compounds described above.

The invention will be more fully understood by reference to thefollowing examples which detail exemplary embodiments of the invention.They should not, however, be construed as limiting the scope of theinvention. All citations throughout the disclosure are hereby expresslyincorporated by reference.

EXAMPLES Preparation of Compounds of Table 1

Experimental Methods:

Unless otherwise specified, all reagents were purchased from commercialsources and were used without further purification. Flash chromatographywas carried out on silica gel (200-400 mesh). ¹H-NMR (300 MHz or 500MHz) and ¹³C-NMR (75 MHz or 125 MHz) spectra were recorded inchloroform-d or DMSO-d6 on a Mercury-300BB or INOVA-500 spectrometer andmass spectra on an Agilent LCMS system.

General Procedure of 6-nitro-quinoxalines 3

A mixture of 4-nitrobenzene-1,2-diamine (10 mmol) and dione 2 (10 mmol)in ethanol (40 ml) was heated at reflux for 24-40 h. The mixture wascooled in an ice bath and the resulting solid was recrystallized frommethanol gave 6-nitro-quinoxalines (3a-d) (85-90%).

General Procedure of 6-amino-quinoxalines 4

6-Nitro-quinoxaline (3, 5 mmol) was hydrogenated in the presence of 5%Pd/C (50 mg) under at room temperature in ethanol (30 ml) for 6-10 h.After the reaction 2 ml DMSO was added to the mixture and filtered. Thefiltrate was poured into water (200 ml) and the precipitate was filteredto give 6-amino-quinoxaline (4a-d) (90-95%).

General Procedure of Quinoxalinylurea and QuinoxalinylthioureaDerivatives 5

To a stirring solution of 4 (1.0 mmol) and triethylamine ordiisopropylethylamine (3.0 mmol) in dichloromethane (20-30 ml), thecorresponding acetyl chloride (1.5 mmol) or phenylisocyanate (1.5-2.0mmol) or toluene sulfonyl chloride (1.5-2.5 mmol) was added. The mixturewas maintained at room temperature for 12-48 h and the reactionmonitored for completion by TLC. Upon consumption of the startingmaterial (4) the solvent was removed and the residue was purified byflash column chromatography on a silica gel to yield the desiredcompounds.

General Procedure of Quinoxalinylurea Derivatives 6

Method A:

To a stirring solution of 4 (1.0 mmol) and diisopropylethylamine (3.0mmol) in dichloromethane (20-30 ml) the corresponding isothiocyanate(1.5-2.0 mmol) was added. The mixture was stirred at room temperaturefor 24-48 h. Upon consumption of the starting material as determined byTLC, the reaction mixture was diluted with 20 mL hexanes. The resultingsolid was filtered and the residue washed with CH₂Cl₂ (2×5 mL). Thematerial was subsequently purified by flash column chromatography on asilica gel to yield the desired compounds.

Method B:

To a stirring solution of 4 (1.0 mmol) and diisopropylethylamine (3.0mmol) in dichloromethane (10-20 mL), triphosgene (0.40 mmol) was added.The mixture was stirred at room temperature for 4-8 h and thecorresponding secondary amine (2.0-3.0 mmol) was added. After stirringfor an additional 8-12 h, the solvent was removed under vacuum and theresidue was purified by flash column chromatography on a silica gelyield the desired compounds.

General Procedure of Quinoxalinylurea Derivatives 7

To a stirring solution of 4 (1.0 mmol) and diisopropylethylamine (3.0mmol) in dichloromethane (20-30 ml) the corresponding isocyanate(1.5-2.0 mmol) was added. The mixture was maintained at room temperaturefor 24-48 h and the reaction monitored for completion by TLC. Uponconsumption of the starting material (4) the reaction mixture wasdiluted with 20 mL hexanes, filtered, the residue washed with CH₂Cl₂(2×5 mL) and purified by flash column chromatography on a silica gel toyield the desired compounds.

Biological Assays of Compounds of Table 1

Cell Growth Inhibition Assay. Human lung A549, human pancreatic AsPC1,human colorectal HT-29, human breast MDA-MB-231, human prostate PC3,human ovarian SKOV3, and human osteosarcoma U2OS tumor cell lines werecultured in basal media containing 10% FBS and maintained in a 37° C.incubator with 5% CO₂. All compounds were screened in 96 well microtiterplate format. In general, each cell line was plated at optimal assaydensity in 95 μl of propagation media in 96 well plates and incubatedovernight. The next day, a standard alamar blue assay was performed (seebelow) on representative wells of each cell line for a T_(z)calculation. Subsequently, cells were treated in duplicate with a 5 μlvolume of either vehicle only, positive controls etoposide and taxol, ortest compounds at 20 μM (highest soluble compound concentration). Thetreated cells were incubated for 72 h and then assayed for growth byalamar blue assay. Briefly, 10 μl of reagent was added to each well andthe plates returned to 37° C. After 3 h, fluorescence at544_(ex)/590_(em) was measured using a SpectraMax M5 (Molecular Devices)plate reader. Growth is represented as a percentage of the control cellsthat were treated with vehicle only and % growth inhibition wasdetermined using the NCI algorithm: T_(z)=number of untreated cells atzero time, C=Number of control cells at 72 h time, and T=number ofcompound treated cells at 72 h time; 100*([T−T_(z)]/[C−T_(z)]).

Caspase 3/7 Activation.

HeLa, MDA-MB-231 (4000 cells/well), and PC3 (2000 cells/well) cells weretreated in 96 well plates with 7c at the indicated times. Caspase Gloreagent (Promega, Inc.) was added and luminescence was measured using aSpectramax M5 (Molecular Devices) plate reader after 1 hr. Rawluminescence values were normalized to alamar blue.

PARP Cleavage.

HeLa cells were treated with 7c for the indicated times. Samples wereprepared by collecting media, trypsinizing cells, and centrifuging toobtain a combined cell pellet from all steps. Cells were lysed in RIPAbuffer and protein content was subjected to SDS-PAGE. PARP cleavage wasdetermined via Western blotting using anti-PARP antibody (Calbiochem#AM30).

Mcl-1 Dependent Apoptosis Assay.

HeLa cells overexpressing the inducible Bcl-xL and Bcl-2 inhibitorBad3SA were induced with doxacyclin (Dox) for 3 hrs. Cells were thentreated with various concentrations of 7c for 12 hrs. Cells were thenfixed and stained with Hoechst dye and the number of condensed nucleiwas counted for each treatment to determine percent death.

Studies on 13-197 (aka 7c)

A cell line designed to specifically monitor the activity of NF-κB inresponse to TNF-α was used for the screen. The compounds were screenedat 20 μM, and their IC₅₀ values determined. The arrow points to thequinoxaline urea analog 13-197 (7c), a quinoxaline urea analog thatinhibits TNF-α mediated NF-κB activation (FIGS. 1A and 1B). Themolecular target of 13-197 in the NF-κB pathway was then determined.

Activation of NF-κB pathway by TNF-α leads to the nuclear translocationof NF-κB (p65). To explore if 13-197 inhibits the nuclear translocationof NF-κB, cells were treated with TNF-α in the presence and absence of13-197. Nuclear NF-κB (p65) levels at indicated time points weredetermined by Western blot analyses. The cells were treated with 20 μMof 13-197 followed by 20 ng/mL of TNF-α. The cell lysates werefractionated at the indicated time points and the nuclear fractions wereprobed for NF-κB levels (p65). The bottom panel shows quantitation(n=3). (FIG. 1C). The results show that 13-197 inhibits TNF-α mediatednuclear translocation of NF-κB and is consistent with inhibition oftranscription (FIG. 1B).

In the resting state IκBα sequesters NF-κB in the cytoplasm by maskingits nuclear localization signal (NLS). Activation of the NF-κB pathwayby TNF-α results in the phosphorylation, ubiquitination and degradationof IκBα, thus unmasking the NF-κB NLS. To determine if 13-197 inhibitsthe phosphorylation of IκBα, cells were treated with and without 13-197followed by TNF-α. The phospho-IκBα levels in the cytoplasm (at theindicated time points) were determined by Western blot analyses. TheNF-κB pathway was activated by TNF-α (20 ng/mL) in cells treated withand without 13-197 (20 μM). Cell lysates were generated at indicatedtimes and probed for p-IκBα, total-IκBα and tubulin. (FIG. 1D). Theresults indicate 13-197 partially inhibits IκBα phosphorylation at theearly time points (5 and 10 min). More importantly, we observe completeinhibition of IκBα phosphorylation at the later time points (60 and 120mins).

IκBα is phosphorylated by IKKβ in response to TNF-α stimulation. TheIκBα-NFκB complex is a better substrate for IKKβ compared to IκBα alone(K_(m)=2.2 μM vs. 1.4 μM and 5-fold change in V_(max)). IκBα is also atarget gene of NF-κB and this serves as one of the feedback mechanism toshut down the NF-κB pathway. The partial inhibition of IκBαphosphorylation at the early time points and the completed inhibition ofthe newly formed IκBα phosphorylation, could be explained if 13-197targets the IκBα-NFκB interface. To test this hypothesis we conductedreciprocal immunoprecipitation (IP) and immunoblotting (IB) at an early(5 min) and a late (120 min) time point post TNF-α stimulation. Celllysates were generated at the indicated times post TNF-α (20 ng/mL)stimulation with and without 13-197 (20 μM). The lysates were subjectedto reciprocal IP-IB with NF-κB (p65) and IκBα antibodies (FIG. 1E). Theresults showed no change in the relative levels of NF-κB pulled down byIκB and vice versa at either time points suggesting 13-197 does notinhibit the IκBα-NFκB complex.

In response to TNF-α stimulation IκBα is phosphorylated by the kinaseIKKβ, therefore we next evaluated if 13-197 inhibits IKKβ. An in vitroELISA assay with full length IKKβ showed that 13-197 inhibits IKKβ withan IC₅₀ value of about 15 μM (FIG. 1F). This indicates that 13-197inhibits IKKβ, however it does not fully explain the results in FIG. 1D.Also as a general rule the IC₅₀ values are lower by at least an order ofmagnitude for in vitro assays compared to cell-based assays. With 13-197we see the comparable effect as the cell-based IC₅₀ value is ˜10 μM(FIG. 1B).

IKKβ is regulated by a plethora of phosphorylation events. TNF-αstimulation induces rapid phosphorylation of Ser177 and Ser181 residuesin the activation loop (T-loop). The T-loop phosphorylated IKKβ is in ahigh-active state and rapidly phosphorylates IκBα (FIG. 1D—5 and 10 minlanes). Concurrently, the T-loop phosphorylated IKKβ undergoesautophosphorylation at C-terminus serine residues which result in alow-active hyperphosphorylated form of IKKβ. If 13-197 preferentiallyinhibits the hyperphosphorylated form of IKKβ it would explain theresults observed in FIG. 1D. This model also agrees with the results inFIG. 1F which shows inhibition of the full length unactivated form ofIKKβ. FIG. 1G pictorially depict a proposed model and a potential targetfor 13-197.

The T-loop phosphorylated form of IKKβ (p-IKKβ) is found in ˜50% ofsurgical tumor specimens and in ˜10% of normal tissues. Also a perfectcorrelation between the presence of TNFα, p-IKKβ and p-IκBα wasobserved. Since NF-κB is constitutively active in pancreatic cancer (PC)we hypothesize that 13-197 will inhibit the constitutively active formof IKKβ in pancreatic cancers. To test this we subjected a panel ofpancreatic cancer cell lines to 13-197 and probed the lysates for p-IκBαlevels (FIGS. 2A and 2B). (A) Pancreatic cancer cell lines were treatedwith 20 μM 13-197 for 2 h and the lysates were probed for p-IκBα. (B)(left) MiaPaCa2 cells were treated with 13-197 (0, 10 and 20 μM) for 2 hand at 20 μM for the indicated times (right). 13-197 robustly inhibitsphosphorylation of IκBα in PC cell lines both in a dose- andtime-dependent manner.

TNF-α stimulation regulates transcription via the p-IKKβ/p-IκBα/NF-κBaxis. It also regulates translation via the p-IKKβ/mTOR/p-S6K/p-eIF4EBPaxis. We speculated that if the IKKβ node is activated in pancreaticcancer we should also see inhibition of p-S6K and p-eIF4EBP. Indeed weobserved decreased p-S6K and p-4EBP1 in MiaPaCa2 cells treated with13-197 (FIG. 2C). (C) MiaPaCa2 cells were treated with 20 μM 13-197 for4 h and 24 h. The cell lysates were probed for p-S6K and p-eIF4EBP1. Insummary the data in FIGS. 2A-C suggests that 13-197 targets aconstitutively active form of IKKβ in pancreatic cancer cell lines (FIG.2D).

We next explored if inhibition of constitutively active IKKβ by 13-197results in the inhibition of pancreatic cancer cell growth. In a typicalexperiment pancreatic cancer cells were subjected to increasing doses of13-197 (FIG. 3A). (A) Pancreatic cells were treated with 13-197 for 72h. IC₅₀ values determined through curve fitting (n=2). In a secondexperiment MiaPaCa2 cells were incubated with 13-197 (at IC₅₀) and thecell growth was monitored over time for 96 h (FIG. 3B). (B) MiaPaCa2cells treated with 11 μM of 13-197 and viability measured at indicatedtimes. *P<0.05, **P<0.005, ***P<0.005. The viability of the cells at theend of these studies was determined by the AlamarBlue dye. The resultsdemonstrate that 13-197 inhibits pancreatic cancer cell growth both in adose- (low-μM IC₅₀ values) and in a time-dependent manner. To determineif the growth inhibition induced by 13-197 is a result of cell cyclearrest, MiaPaCa2 cells were treated with 13-197 and subjected cell cycleanalysis. Cells treated with 13-197 arrested in the G1 phase (FIG. 3C).(C) MiaPaCa2 cells were treated with 11 μM 13-197 for 24 h followed bycell cycle analyses (n=3). Consistent with the G1 arrest we observereduced levels of the corresponding cell cycle markers E2F, PCNA andCyclin D1 (FIG. 3D). Together the data in FIGS. 3A-D demonstrates that13-197 inhibits pancreatic cancer cell growth by inducing G1 arrest ofcells.

Next we probed the effect of 13-197 on apoptosis related proteins. Mcl-1and Bcl-xL are antiapoptotic proteins that sequester the proapoptoticproteins (Bax/Bak). XIAP and Survivin belong to the IAP family and areinhibitors of caspase activation. (E) Dose- and time-dependent effectsof 13-197 on the levels of anti apoptotic proteins Mcl-1, Bcl-xL, XIAPand Survivin in MiaPaCa2 cells. MiaPaCa2 cells were treated with 13-197at the indicated doses 2 h (left panel). Cells were treated with 20 μMthe cell lysates were generated probed at the indicated time points(right panel). The dose response study (2 h) shows a significantdecrease in the Mcl-1 levels compared to the other proteins (FIG. 3E,left panel). However, longer incubation times at 20 μM shows a decreasein all proteins (FIG. 3E, right panel). The distinct kinetics (Mcl-1 vsBcl-xL/XIAP/Survivin) suggests that Mcl-1 could be down regulated byIKKβ directly while the others are down regulated by the inhibition ofNF-κB activation. In all the data shows that 13-197 down regulatesantiapoptotic proteins both in a dose- and time-dependent manner.

MiaPaCa2 cells were subjected to 11 μM 13-197 for 24 h and apoptosis wasmeasured by the live/dead cellular assay. Cells treated with 13-197showed a 4-fold increase in percentage of apoptotic cells when comparedto untreated cells (FIG. 3F). This is consistent with the downregulation of antiapoptotic proteins (FIG. 3E).

NF-κB regulates the expression of several genes such as IL-8, VEGF,ICAM1 and MMP-9 that are implicated in angiogenesis, invasion andmetastasis. Invasion of cells through matrigel coated microporouspolycarbonate membrane was measured in the presence and absence of13-197 (n=3). Transwell serum driven migration of cells in the presenceand absence of 13-197 was measured after a 24 h incubation. (n=3).**P<0.005 and ***P<0.0005. We observed that 13-197 inhibited both theinvasion and migration of MiaPaCa2 cells by about 50% (FIG. 3G). Thissuggests that 13-197 has the potential to not only inhibit growth oftumors but also inhibit metastasis.

In summary cellular studies with 13-197 suggests that it targets the“disease state” of IKKβ (T-loop and C-terminus phosphorylated). Inpancreatic cancer cells 13-197 targets the constitutively activated IKKβas it down regulates transcription via the IKKβ/IκBα/NF-κB axis andperturbs translation through the IKKβ/mTOR/S6K-eIF4EBP axis. 13-197 alsodown regulates antiapoptotic proteins. As a consequence 13-197 inhibitscell growth and induces apoptosis in MiaPaCa2 cells.

PK difficulties account for more than 50% of drug development failurespreventing new chemical entities (NCEs) from reaching the market,whereas toxicity issues and lack of efficacy account for only 30% ofdevelopment failures. As a result, in addition to paying attention tothe traditional concern of attaining potency and selectivity towards thebiological target of interest. PK considerations have been moved toearly stages of drug discovery, a significant paradigm shift in thepharmaceutical industry. Along these lines we determined the PKparameters for our NCE 13-197.

Lipinski's rule of five serves as a guide to determine if compounds willbe orally bioavailable. Analysis of 13-197 suggested that it would beorally available. 13-197 was formulated in cremaphor EL a commonly usedexcipient in drugs for pharmacokinetic (PK) and tissue distributionstudies. A mass spectrometry method was established to determine 13-197levels in plasma and tissue. Mice were dosed at 150 mg/Kg orally andsacrificed at indicated time points. 13-197 levels in blood and varioustissue samples were determined by mass spectrometry (FIGS. 4A and 4B).The PK properties of 13-197 are described as an inset in FIG. 4A and thetissue levels as an inset in FIG. 4B. The results show that 13-197 isorally available, has an excellent distribution (large V_(d)) and isprimarily cleared through the liver and the kidney.

Once we established that 13-197 is orally available, MiaPaca2 cells wereorthotopically placed in the pancreas of nude mice. The mice wereallowed to heal after surgery and the tumors were allowed to grow for 2weeks at which time they were palpable. The tumor bearing mice wererandomized and half the animals were treated orally with 13-197 at 150mg/Kg in cremaphor daily. At the end of 30 days the mice were sacrificedand the tumors weights and volumes were measured (FIG. 4C). We observedabout 50% reduction in both the tumor weight and volume in the 13-197treated animals compared to vehicle treated animals. We also found fewertumor nodules in other organs of 13-197 treated animals compared tovehicle treated animals (FIG. 4D). These result are consistent with ourcellular data suggest inhibition of tumor growth and metastases by13-197. Proliferation index and microvessel density are measures of thenumber of cells dividing in the tumor and angiogenesis indicatorsrespectively. We observe a reduction of both in 13-197 treated tumorscompared to vehicle controls (FIGS. 4E and 4F). The tumor tissue wasscored and the results indicate decreased inflammation and increasednecrosis in the 13-197 treated mice (FIG. 4G). These results areconsistent with the inhibition of NF-κB. We also determined the 13-197levels in the pancreas, liver and serum at the end of the study (FIG.4H). Consistent with our PK data the highest drug levels found in theliver. Since 13-197 is primarily cleared by the liver we measured thelevels of aspartate aminotransferase (AST) and alanine aminotransferase(ALT) which are biochemical markers of the liver toxicity in 13-197 andvehicle treated mice. We did not observe a difference in the ALT or ASTlevels in 13-197 treated mice when compared vehicle treated miceindicating the absence of liver toxicity (FIG. 4I). We also conducted amacroscopic examination of the organs and found no obvious toxicity inthe 13-197 treated animals. In summary our studies in animals show that13-197 is orally available with excellent distribution (high V_(d)). Itinhibits tumor growth and metastasis in mice bearing pancreatic tumorswith no obvious toxicity.

The effect of 13-197 on the IkBα phosphorylation in therapy-resistantMCL cells was assessed. 5×10⁶ each MCL cells were cultured in RPMI mediacontaining vehicle (DMSO) or indicated concentration of 13-197 fordifferent time point (2-24 hours). After time incubation, cells wereharvested and whole cell lysate was prepared. 20-50 μg of total proteinfrom whole cell lysate was subjected to western blot for the expressionof IκBα phosphorylation. FIG. 7A shows western blot results for IkBαphosphorylation by 13-197 in Granta-519 parental (GP) cells in adose-dependent manner. FIG. 7B indicates phosphorylation status of IkBαby 13-197 in GP, GRL, GRK and GRR MCL cells in time- and dose-dependentmanner. Total IkBα was used as an internal control in these experiments.

The effect of 13-197 on the down-regulation of the NF-κB (p65)phosphorylation and their nuclear translocation in the therapy-resistantMCL cell lines was assessed. 5×10⁶ each MCL cells were cultured in RPMImedia containing vehicle (DMSO) or indicated concentrations (1-100 μM)of 13-197 for 1 or 2 hours. After time incubation, cells were harvestedand whole cell lysate or cytoplasmic/nuclear fraction was prepared.20-50 μg of total protein from whole cell lysate or cytoplasmic/nuclearfractionated lysate was subjected to western blot for the expression ofNF-κB (p65) phosphorylation and their nuclear translocation. FIG. 8Ashows phosphorylation status of NF-κB (p65) by 13-197 in Granta-519cells in a time-dependent fashion. FIG. 8B shows western blot resultsfor NF-κB nuclear translocation by 13-197 in Granta-519 MCL cells in adose-dependent manner after cytoplasmic and nuclear fractionation of thecells. PARP-1 and α-tubulin were also detected to confirm cytoplasmicand nuclear fractionation of proteins.

The effect of 13-197 on the down-regulation of the NF-κB target in thetherapy-resistant MCL cells was assessed. 5×10⁶ each MCL cells werecultured in RPMI media containing vehicle (DMSO) or indicatedconcentration of 13-197 for 24 hours. After incubation time, cells wereharvested and whole cell lysate was prepared. ˜50 μg of total proteinfrom whole cell lysate was subjected to western blot for the expressionof NF-κB target molecules. FIG. 9A shows status of IkBα phosphorylation,MCL-1 and BCL-XL in 13-197 (10 μM) treated-Granta-519 (GP), -GRL, -GRKand -GRR MCL cells. FIG. 9B represents expression level of cyclin D1 in13-197 treated-Granta-519 (GP) and GRL MCL cells in a dose-dependentmanner. β-actin was used as an internal control in the each experiment.

The effect of 13-197 on the down-regulation of the mTOR target moleculesin the therapy-resistant MCL cells was assessed. 5×10⁶ different MCLcells were cultured in RPMI media containing vehicle (DMSO) or 13-197(10 μM) for 24 hours. After incubation time, cells were harvested andwhole cell lysate was prepared. 50 μg of total protein from whole celllysate was subjected to western blot for the expression ofphosphorylation status of mTOR pathway molecules include S6K and 4E-BP1.β-actin was used as an internal control in the experiment. GP indicates‘Granta-519’. The results are shown in FIG. 10

The effect of 13-197 on therapy-resistant MCL cells growth/proliferationin vitro was assessed. Therapy-resistant MCL cells [Granta-519 parental(GP); GRL, derived from liver; GRK, derived from kidney and GRR, derivedfrom lungs] MCL cells (10,000) were cultured in RPMI media containing0.5, 1.0, 5.0, 10 and 20 μM 13-197 compound in 96-well plates for 48hours. FIG. 11A-11D: MTT reagent was added 2 hours before cell harvest.MTT developed color was read using a microplate reader at 570 nm Foldchange was calculated with respect to absorbance exhibited by vehicle(DMSO)-treated cells. The values represent the means±SD from four wellsof at least three independent experiments. FIG. 11E-11H: ³[H]-thymidine(0.5 μCi) was added 15 hours before harvesting the cells, andincorporated radioactivity was determined using a scintillation counter.The values represent the means±SD from triplicate wells of at leastthree independent experiments.

The effect of 13-197 on MCL cells apoptosis was assessed. AnnexinV-FITCapoptosis detection assay was used to access percent of cells undergoingapoptosis in granta 519 parental, GRL, GRK and GRR MCL cell linesfollowing treatment with 10 μM 13-197 for 48 hours. The percent of cellsundergoing apoptosis indicate annexin and propidium iodide (PI)positive, as shown in FIG. 12. The values represent the means±SD ofthree separate experiments.

The cytomorphology of Wright-Giemsa stained MCL cells followingtreatment with 13-197 was assessed. Exponentially growingtherapy-resistant MCL cells were treated with 13-197 (10 μM) for 48 and72 hours. After treatment incubation cells were processed for Giemsastaining followed by cyto-spin. The cells were observed under the brightfield microscope and images were captured at 40× magnification, as shownin FIG. 13.

REFERENCES

-   Delhase, M., Hayakawa, M., Chen, Y. & Karin, M. Positive and    negative regulation of IkappaB kinase activity through IKKbeta    subunit phosphorylation. Science 284, 309-313 (1999).-   Hacker, H. & Karin, M. Regulation and function of IKK and    IKK-related kinases. Sci STKE 2006, rel3 (2006).-   Chiang, C. W., Liu, W. K., Chiang, C. W. & Chou, C. K.    Phosphorylation-dependent association of the G4-1/G5PR regulatory    subunit with IKKbeta negatively modulates NF-kappaB activation    through recruitment of protein phosphatase 5. Biochem J 433, 187-196    (2011).-   Lee, D. F. et al. IKK beta suppression of TSC1 links inflammation    and tumor angiogenesis via the mTOR pathway. Cell 130, 440-455    (2007).-   Bjellqvist, B. et al. The focusing positions of polypeptides in    immobilized pH gradients can be predicted from their amino acid    sequences. Electrophoresis 14, 1023-1031 (1993).-   Druker, B. J. et al. Effects of a selective inhibitor of the Abl    tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2,    561-566 (1996).-   Sen, R. & Baltimore, D. Inducibility of kappa immunoglobulin    enhancer-binding protein Nf-kappa B by a posttranslational    mechanism. Cell 47, 921-928 (1986).-   Baldwin, A. S., Jr. The NF-kappa B and I kappa B proteins: new    discoveries and insights. Anna Rev Immunol 14, 649-683 (1996).-   Miyamoto, S., Chiao, P. J. & Verma, I. M. Enhanced I kappa B alpha    degradation is responsible for constitutive NF-kappa B activity in    mature murine B-cell lines. Mol Cell Biol 14, 3276-3282 (1994).-   Conti, L. et al. Induction of relA(p65) and I kappa B alpha subunit    expression during differentiation of human peripheral blood    monocytes to macrophages. Cell Growth Differ 8, 435-442 (1997).-   Farrow, B. & Evers, B. M. Inflammation and the development of    pancreatic cancer. Surg Oncol 10, 153-169 (2002).-   Aggarwal, B. B. Nuclear factor-kappaB: the enemy within. Cancer Cell    6, 203-208 (2004).-   Wang, W. et al. The nuclear factor-kappa B RelA transcription factor    is constitutively activated in human pancreatic adenocarcinoma    cells. Clin Cancer Res 5, 119-127 (1999).-   Chandler, N. M., Canete, J. J. & Callery, M. P. Increased expression    of NF-kappa B subunits in human pancreatic cancer cells. J Surg Res    118, 9-14 (2004).-   Weichert, W. et al. High expression of RelA/p65 is associated with    activation of nuclear factor-kappaB-dependent signaling in    pancreatic cancer and marks a patient population with poor    prognosis. Br J Cancer 97. 523-530 (2007).-   Cascinu, S. et al. COX-2 and NF-κB overexpression is common in    pancreatic cancer but does not predict for COX-2 inhibitors activity    in combination with gemcitabine and oxaliplatin. Am J Clin Oncol 30,    526-530 (2007).-   Pan, X. et al. Nuclear factor-kappaB p65/relA silencing induces    apoptosis and increases gemcitabine effectiveness in a subset of    pancreatic cancer cells. Clin Cancer Res 14, 8143-8151 (2008).-   Kong, R. et al. Downregulation of nuclear factor-kappaB p65 subunit    by small interfering RNA synergizes with gemcitabine to inhibit the    growth of pancreatic cancer. Cancer Lett 291, 90-98 (2010).-   Fujioka, S. et al. Function of nuclear factor kappaB in pancreatic    cancer metastasis. Clin Cancer Res 9, 346-354 (2003).-   Mercurio, F. et al. IKK-1 and IKK-2: cytokine-activated IkappaB    kinases essential for NF-kappaB activation. Science 278, 860-866    (1997).-   Zandi, E., Chen, Y. & Karin, M. Direct phosphorylation of IkappaB by    IKKalpha and IKKbeta: discrimination between free and    NF-kappaB-bound substrate. Science 281, 1360-1363 (1998).-   Darzynkiewicz, Z., Gong, J., Juan, G., Ardelt, B. & Traganos, F.    Cytometry of cyclin proteins. Cytometry 25, 1-13 (1996).-   Salvesen, G. S. & Abrams, J. M. Caspase activation—stepping on the    gas or releasing the brakes? Lessons from humans and flies. Oncogene    23, 2774-2784 (2004).-   Gilmore, T. D. Introduction to NF-kappaB: players, pathways,    perspectives. Oncogene 25, 6680-6684 (2006).-   Cheng, A. et al. Computation of the physio-chemical properties and    data mining of large molecular collections. J Comput Chem 23,    172-183 (2002).-   Clark, D. E. & Grootenhuis, P. D. Progress in computational methods    for the prediction of ADMET properties. Curr Opin Drug Discov Devel    5, 382-390 (2002).-   Lipinski, C. A., Lombardo. F., Dominy, B. W. & Feeney, P. J.    Experimental and computational approaches to estimate solubility and    permeability in drug discovery and development settings. Adv Drug    Deliv Rev 46, 3-26 (2001),-   Morikane, K. et al. Organ-specific pancreatic tumor growth    properties and tumor immunity. Cancer Immunol Immunother 47, 287-296    (1999).-   van Diest, P. J., van der Wall, E. & Baak, J. P. Prognostic value of    proliferation in invasive breast cancer: a review. J Clin Pathol 57,    675-681 (2004).-   Weidner, N. Current pathologic methods for measuring intratumoral    microvessel density within breast carcinoma and other solid tumors.    Breast Cancer Res Treat 36, 169-180 (1995).-   De Ritis, F., Coltorti, M. & Giusti, G. An enzymic test for the    diagnosis of viral hepatitis; the transaminase serum activities.    Clin Chim Acta 2, 70-74 (1957).-   Chen, Q. et al. 2,3-Substituted quinoxalin-6-amine analogs as    antiproliferatives: a structure-activity relationship study. Bioorg    Med Chem Lett 21, 1929-1932 (2011).

What is claimed:
 1. A compound having a structure selected from:

or a salt thereof.
 2. The compound of claim 1, having a structure

or a salt thereof.
 3. The compound of claim 1, having a structure

or a salt thereof.
 4. The compound of claim 1, having a structure

or a salt thereof.
 5. The compound of claim 1, having a structure

or a salt thereof.
 6. The compound of claim 1, having a structure

or a salt thereof.
 7. The compound of claim 1, having a structure

or a salt thereof.
 8. A method of decreasing the inhibitor of kappa Bkinase β activity in a cell, comprising contacting the cell with aneffective amount of the compound of claim
 1. 9. The method of claim 8,wherein the inhibitor of kappa B kinase β is hyperphosphorylated.
 10. Amethod of inhibiting the nuclear factor kappaB signaling pathway in acell, comprising contacting the cell with an effective amount of thecompound of claim
 1. 11. A method of inhibiting the mammalian target ofrapamycin signaling pathway in a cell, comprising contacting the cellwith an effective amount of the compound of claim 1.